Blood vessel-growth promoter

ABSTRACT

A neo-vascularization promoter containing acetylsalicylic acid or its pharmaceutically acceptable salt as an active ingredient.

TECHNICAL FIELD

The present invention relates to a preparation containingacetylsalicylic acid as an active ingredient having excellentneo-vascularization promoting activity, and a method for treating apatient suffering from a disease caused by lack of neo-vascularizationwhich comprises administering said preparation.

BACKGROUND ART

Neo-angiogenesis is a phenomenon that a small blood vessel is newlyformed from an already existed blood vessel. The prognosis of thecircular diseases such as myocardic infarction is affected by manyfactors, and the degree of the development of collateral vessels isconsidered to be one of the most important determining-factors of theconvalescence. When collateral vessels are sufficiently developed, evenif arctation or infarction occurs, ischemia or necrosis of the tissue isinhibited, and size of the infarction decreases or the convalescence isimproved.

From of old as a mechanism of collateral vessel-formation, change of theinner pressure of the blood vessel and hemokinesis (blood flow) has beenmade a big point, but it was reported that in vascular endothelial cellsand vascular smooth muscular cells, cytodieresis figure associated withDNA synthesis was recognized in case of formation of collateral vessel.Therefore, it becomes to be recognized that the process of collateralvessel-formation is not only expansion due to physical factors of thealready existed inosculated vessel, but also at least a part of theprocess is neo-vascularization which proliferation of cells composingblood vessel wall participates.

On the other hand, neo-vascularization is widely confirmed in thephysiological phenomenon such as wound healing. Its mechanism isconsidered to be due to migration or proliferation of vascularendothelial cells from venula or capillary which is already existed, ordue to tube formation. Furthermore, interactions via cytokine or growthfactor among other cells (fibroblast, smooth muscular cells) surroundingvascular endothelial cells is also considered to be an important factorin regard to wound healing.

Nowadays, on substances having neo-vascularization promoting activitysuch as fibroblast growth factor (FGF), hepatocyto growth factor (HGF),etc. and neo-vascularization promoting factor such as vascularendothelial cell growth factor (VEGF), etc., had been evaluated thepossibility as an agent for treating an angiogenic disease, and theresults were reported (See Internal medicine, Vol. 85, No. 5. page893-897 (May, 2000), Internal medicine, Vol. 85, No. 5. page 886-892(May, 2000), etc.). However, these substances have not been in practiceand the development of the excellent substances is still desired.

DISCLOSURE OF INVENTION

The object of the present invention is to provide an agent for treatinga disease caused by lack of neo-vascularization having excellentneo-vascularization promoting activity with less side effect.

The present invention relates to a neo-vascularization promoter(hereinafter abbreviate as a neo-vascularization promoter of the presentinvention or a drug of the present invention.) containingacetylsalicylic acid or its pharmaceutically acceptable salt as anactive ingredient.

The present invention relates to a neo-vascularization promotercontaining an amount of 0.01 to 80% by weight of acetylsalicylic acid orits pharmaceutically acceptable salt.

The present invention relates to a method for treating a patientsuffering from a disease caused by lack of neo-vascularization diseasewhich comprises administering said preparation to the patient in aneffective amount of it.

The present inventors have extensively studied to dissolve the aboveproblems, and have found that acetylsalicylic acid or itspharmaceutically acceptable salt unexpectedly showed strongneo-vascularization promoting activity. Thus the present invention wascompleted.

BEST MODE FOR CARRYING OUT THE INVENTION

The present inventors confirmed that proliferation activity of humanumbilical vein endothelial cells (HUVEC) was shown by addingacetylsalicylic acid to HUVEC in vitro test.

Furthermore, in vitro test using a mixture of human vascular endothelialcell and fibroblast by adding acetylsalicylic acid thereto, promotingactivity of tube formation was shown and further in vivo test onneo-vascularization by using rats, neo-vascularization promotingactivity was shown by administration of acetylsalicylic acid.

This effect depends on the concentration of acetylsalicylic acid in apreparation, but the efficacy is not changed even beyond a certainamount.

The neo-vascularization promoter of the present invention is usuallyadministered systematically, locally, orally or parenterally.

The amount and administration route of the neo-vascularization promoterof the present invention are not specially limited, but the mostsuitable amount is determined in accordance with the administrationroute, ages, sex, severity, and frequency, body weight, etc.

In case of the systemic application such as injection, oral applicationor insertion of suppository, the amount is 0.01 to 4.5 g/adult/day onceor more times a day.

As the neo-vascularization promoter of the present invention,acetylsalicylic acid or its pharmaceutically acceptable salt itself maybe used, but a preparation prepared by blending it with fillers used inusual preparation, or other additives may be used.

The blood concentration of acetylsalicylic acid contained in thepreparation of the present invention is 1 ng/mL to 550 μg/mL (0.0005 to3000 μM), preferably 10 ng/mL to 55 μg/mL, more preferably 100 ng/mL to25 μg/mL. When the concentration of acetylsalicylic acid is less than 1ng/mL, the effect is not enough, and when beyond 550 μg/mL, there is ahigh possibility to occur side effect.

The tissue concentration of acetylsalicylic acid contained in thepreparation of the present invention is 0.01 ng/g to 1000 μg/g,preferably 0.1 ng/g to 500 μg/g, more preferably 1 ng/g to 100 μg/g.When the tissue concentration of acetylsalicylic acid is less than 0.01ng/g, the effect is not enough, and when beyond 1000 μg/g, there is ahigh possibility to occur side effect.

The acetylsalicylic acid as an active ingredient of the presentinvention is a pharmaceutically acceptable amino acid salt such aslysine salt or an inorganic salt such as sodium salt as well asacetylsalicylic acid.

The preparation of the present invention is any preparation such as asolid preparation, a liquid composition or other composition suitablefor oral administration, parenteral application such as injection,suppository, and if necessary a suitable preparation is selected.

As an oral preparation, tablets, pills, capsules, powders, granules,solutions, etc. can be illustrated.

On the other hand, as an external preparation, as long as thepreparation can be applied directly to the local surface of the skin, itis not specifically limited, such as preparation such as ointments,creams, gels, patches, solutions (suspensions, emulsions, lotions,etc.), cataplasms, tapes, external powders, aerosols, etc.

The amount of acetylsalicylic acid or its pharmaceutically acceptablesalt contained in the preparation depends on the form of thepreparation, but the amount is 0.005 to 80% by weight per total weightwhich shows enough effect, preferably 0.01 to 70% by weight, morepreferably 0.01 to 50% by weight. When the amount of acetylsalicylicacid contained therein is less than 0.005% by weight, it is notpreferable because the activity of acetylsalicylic acid is not shownenough, and when beyond 80% by weight, it is difficult to prepare thepreparation.

The diseases directed to the present invention is neo-vascularizationtogether with repairing of the tissue, for example, temperaturedisturbance such as fire injury, heat injury, heat injured ulcus,frostbite, etc.; external injury such fracture, abrasion, incise wound,bite, acne, bite, etc.; blood vessel or lymphotube injury such as Bürgerdisease, lymphedemas, crus ulcer, etc.; postoperative wound, such asdonor site, sutural etc.; dermal wound such as decubitus, compressiveulcer, diabetic ulcer-gangrene, stoma, radiation injury, chemicalinjury, etc.; dermal injury such as blister, erosion etc.; ischemicdisease (circular disease), such as myocardiac infarction, etc.

EXAMPLE

The present invention is explained by test-examples andpreparation-examples by using acetylsalicylic acid, but the presentinvention should not be limited by these examples.

Test

The neo-vascularization promoting activity of the present invention wasevaluated in vitro test.

Test 1

Proliferation Test on Human Umbilical Vein Endothelial Cells (HUVEC)

In a plate having 96 wells a certain amount (2000˜4000 cells) of HUVECwere seeded, and the cells were cultured in the CS-C broth containing 5%fetal bovine serum (Cell Systems Co.) over night to adhere thereto.After the culture broth was filtered by suction, to a minimum essentialmedium (MEM) broth containing 1% FBS, acetylsalicylic acid was added ata concentration (0, 0.055, 0.55, 5.5, 55, 555 μM, respectively) and eachmixture was cultivated. After 0, 24 and 48 hours the number of alivecells was counted. As a positive control vascular endothelial growthfactor (VEGF; Vascular Endothelial Growth Factor-A) (20 ng/mL) was used.In this measurement, Cell Counting Kit-8 (Dojin Chemical Co.) was used.

The result was shown in Table 1. TABLE 1 Change of the number of HUVECRelative absorvancy Conc. of drug After After After Group (drug) (μM) 0hour 24 hours 48 hours Control — 1.000 1.236 1.200 VEGF 20 ng/mL 1.0032.041 1.889 Acetylsalicylic acid 0.055 1.102 1.252 1.303 0.55 1.1442.001 1.709 5.5 1.084 1.811 1.575 55 1.107 1.754 1.475 555 1.017 1.0440.699

From the result shown in Table 1, when acetylsalicylic acid was added inmore than 0.055 μM, proliferation promoting activity of HUVEC wasconfirmed and this effect was nearly equal to the effect of vascularendothelial growth factor (VEGF). When acetylsalicylic acid was added inthe amount of more than 555 μM, there showed the tendency that itsproliferation promoting effect was not recognized or was even depressed.

Test 2

Measurement of Neo-Vascularization Ability

The tube formation by a mixture-system of human vascular endothelialcells (HUVEC) and fibroblast was investigated by CD31 staining kit(Kurabo Ind.) to be visualized, and be made score by an analytical soft(NIH Image). A broth containing acetylsalicylic (0.5, 5.5, 55 μM;changed every three days) was added to neo-vascularization kit(Angiogenesis Kit KZ-1000; Kurabo Ind.) prepared by mixing humanvascular endothelial cells and fibroblast, and the resulting broth wascultivated for 11 days. Then immuno histochemical study was performed byusing CD31 staining kit, and the color photo was taken. The picture wasanalyzed using NIH Image and the amount of the formed tubes was scoredas an area to evaluate. As a positive control, vascula endothelialgrowth factor (VEGF; 10 ng/mL) and as a negative control sramine (100μM) were used, respectively.

The result was shown in Table 2. TABLE 2 Neo-vascularization abilityConc. of drug Relative area Group (μM) After 11 days Control — 1.00 VEGF10 ng/mL 1.71 Sramine 100 0.04 Acetylsalicylic acid 5.5 1.17 55 1.08

From the result shown in Table 2, the group containing acetylsalicylicacid showed tube-formation promoting activity.

Test Example 3

Neo-Vascularization Test Using Rats

Wister female rats (7 weeks old; n=6) were anesthetized with ether, anda urethane sponge (diameter: 8 mm) previously permeating a drug wasinserted in subcutaneous tissue of right shoulder. As a test drug,acetylsalicylic acid (2 mg) and as a positive control retinoid (0.5 mg)were administered, respectively. Four days later after insertion of thesponge, 5% gelatin solution containing 5% carmine dye kept at 37° C. wasadministered under ether anesthesia in tail vein of the rat. Afterinjection, the rats in suspended animation were left at 4° C. for 2hours, and the gelatin solution containing carmine dye distributed inblood vessel was solidified. After butcher of the rats, the sponge withgranulation tissue was extracted and the dye therein was extracted.Absorbancy at 530 nm was measured to calculate the amount of the carminedye.

The result was shown in Table 3. TABLE 3 Amount of carmine dye Testgroup Carmine dye (μg/mL) Acetylsalicylic acid 16.0 ± 4.2 Retinoid 27.1± 6.8 Control  9.2 ± 5.7

From the result shown in Table 3, as the amount of carmine dye was muchmore than control, it was confirmed that neo-vascularization waspromoted.

As mentioned above, in the test in vitro using HUVEC, etc., the effectof acetylsalicylic acid on cell proliferation and tube-formation wastested, and as a result proliferation and tube-formed activity of HUVECwas confirmed. Therefore, it was found that the preparation of thepresent invention was very useful as a neo-vascularization promoter.

Preparation Example 1

Tablets

Acetylsalicylic acid (100 parts by weight), lactose (200 parts byweight), starch (50 parts by weight), crystalline cellulose (147 partsby weight) and magnesium stearate (3 parts by weight) were blended andthe mixture was tableted to prepare tablets weighing 100 mg per tabletand containing acetylsalicylic acid 20 mg per tablet.

Preparation Example 2

Capsules

Acetylsalicylic acid (200 mg), microcrystalline cellulose (400 mg),silicon dioxide (10 mg) and magnesium stearate (5 mg) were added in hardcapsules to prepare capsules.

Preparation Example 3

Suppositories

Sucrose fatty acid ester (50 mg) was dispersed in a part of hard fat andthe mixture was melted by heating at more than 90° C. Afteracetylsalicylic acid (750 mg) and the rest of hard fat were dispersed at60° C., the mixture was well blended with the previously preparedsolution. The mixture was filled in a vessel for suppositories andcooled to solid to give suppositories of total weight 2500 mg.

Preparation Example 4

Ointments

Hydrocarbon gel (93 parts by weight) and isopropyl adipate (5 parts byweight) were mixed under heating, and thereto was added acetylsalicylicacid (2 parts by weight). The mixture was well kneaded under agitatingto prepare ointments.

Preparation Example 5

Tapes

Stylene-isoprene-stylene block copolymer (30 parts by weight),hydrogenated rosin glycerin ester (25 parts by weight), polybutene (9parts by weight) and dibutyl hydroxytoluene (1 part by weight) were putin a kneader under warming, and the mixture was stirred under warming tomelt it. Separately, acetylsalicylic acid (10 parts by weight),isopropyl myristate (10 parts by weight) and hydrogenated rosin glycerinester (15 parts by weight) were mixed and stirred, and the mixture wasadded to the mixture previously prepared. The mixture was completelykneaded and the ointment base was spread on cloth to cut in desired sizeto prepare tapes.

Preparation Example 6

Powders

Potato starch (76 parts by weight), zinc oxide (4 parts by weight) andacetylsalicylic acid (20 parts by weight) were mixed until it becamecompletely homogenously to prepare powders.

INDUSTRIAL APPLICABILITY

It is expected that the neo-vascularization promoter of the presentinvention containing acetylsalicylic acid or its pharmaceuticallyacceptable salt as an active ingredient shows excellent therapeuticeffect against, angiogenic diseases required for neo-vascularization(temperature disturbance such as fire injury, heat injury, heat injuredulcus, frostbite, etc.; external injury such fracture, abrasion, incisewound, bite, acne, bite, etc.; blood vessel or lymphotube injury such asBürger disease, lymphedemas, crus ulcer, etc.; postoperative wound, suchas donor site, sutural, etc.; dermal wound such as decubitus,compressive ulcer, diabetic ulcer-gangrene, stoma, radiation injury,chemical injury, etc.; dermal injury such as blister, erosion, etc.;ischemic disease (circular disease) such as myocardiac infarction,etc.).

1. A neo-vascularization promoter containing acetylsalicylic acid or itspharmaceutically acceptable salt as an active ingredient.
 2. Theneo-vascularization promoter according to claim 1 whereinacetylsalicylic acid or its pharmaceutically acceptable salt iscontained in an amount of 0.01 to 80% by weight.
 3. A method forpromoting neo-vascularization administering to a patient suffering fromdisease caused by lack of neo-vascularization an effect amount ofacetylsalicylic acid or its pharmaceutically acceptable salt.